Method for assay of antibodies and antibody assay device

ABSTRACT

The invention relates to a technology by which antibodies directed to sources of infection in body fluids can be assayed with high accuracy, expediency and specificity. More particularly, the invention provides an antibody immunoassay method in which the antigen-antibody reaction between a target antibody in a sample and an assay antigen is conducted in the presence of an  E. coli  component and an antibody assay method which comprises using a reagent having a specific affinity for the Fc region of an antibody IgG as the antibody assay reagent. The invention further provides an antibody assay device comprising a solid-phase support having at least (a) a first region to which a sample is applied and (b) a second region where the antibody in the sample is reacted as disposed in such an arrangement that the sample is wicked from the first region to the second region and a labeling means for detection of the result of the reaction in the second region, characterized in that the (b) second region is provided with (i) a test site in which a ligand for capturing the target antibody to be assayed is immobilized and (ii) a control site in which a ligand for capturing an arbitrary antibody occurring in the sample is immobilized.

TECHNICAL FIELD

[0001] The present invention relates to a method of detecting orquantitating antibodies in samples and more particularly to a method bywhich antibodies against sources of infection such as bacteria andviruses as occurring in clinical body fluid samples, particularly urinesamples, can be detected or assayed with high accuracy, expediently, andwith good specificity.

[0002] The present invention in a further aspect relates to a device fordetecting or quantitating an antibody in a sample and more particularlyto a device with which the antibody against a source of infection asoccurring in clinical body fluid samples, particularly urine samples,can be detected or assayed with high accuracy, expediently, and withgood specificity.

[0003] The invention further relates to an antibody assay reagent kitwhich is useful for the above antibody assay method and the assay methodusing said antibody assay device.

BACKGROUND ART

[0004] Detection of antibodies specific to various sources of infection(pathogens) such as bacteria and viruses, which may occur in bodyfluids, is a useful indirect means for the diagnosis of an infection.Therefore, immunological assay techniques and devices designed to detectan antibody by utilizing a pathogen or a component of the pathogen as anassay antigen have heretofore been used in a broad field of diagnosis.

[0005] Such an immunoassay method using a pathogen or a componentthereof as an assay antigen is advantageous in that the necessary assaysystem can be easily established but is not fully satisfactory insensitivity and specificity, thus leaving room for improvement.

[0006] As an immunoassay device for use in such immunological assays,there can be mentioned a strip of porous material on which a bindingassay (antigen-antibody reaction) is carried out. An assay device ofthis type takes advantage of the capillary property of a poroussubstrate, that is to say a body fluid applied to one end of a porousstrip migrates toward the other end. Thus, when a test sample (liquid)containing a substance to be assayed is applied to one end of the stripcarrying various reagents disposed successively in strategicalpositions, the sample migrates by capillary action along the strip andencounters those reagents in said positions in succession to undergoreactions. The existence of the substance to be assayed can be confirmedand its amount be determined by detecting a signal from the detectablelabel included in the ligand-receptor coupling system.

[0007] The immunoassay technique utilizing the above principle is oftencalled immunocapillary assay or imunochromatographic assay, and has beendescribed in WO No. 87/02774, EP No. 0306772 and other publications. Asto modifications of the technique, the inventions described in JapaneseUnexamined Patent Publication NO. 63865/1989, Japanese Unexamined PatentPublication NO. 299464/1989 and Japanese Unexamined Patent PublicationNO. 167497/1994 can be mentioned.

[0008] The above-mentioned device is advantageous in that no specificinstrument is required for determination and the assay can be completedeasily and within a short time but have room for improvement insensitivity and specificity.

[0009] In addition, because the device performs one test only, anegative or positive control sample cannot be concurrently determined,with the consequent disadvantage that it is impossible to judge whetherthe result is a reliable data generated by the proper determination.

[0010] Generally speaking, urine and saliva, among body fluids, arefavored as clinical test samples because its collection requires noinvasive procedure and is easy and safe as compared with blood.

[0011] However, it is usual that the concentrations of antibodiespresent in such samples are extremely low, for example of the order ofone-thousandth to one-ten thousandth of the concentrations in blood. Inaddition, urine samples collected from subjects who have taken largequantities of water are extremely thin, with the result that a largevariation is inevitable in antibody titer among samples.

[0012] In such cases, with the conventional assay device describedabove, the test will be negative when the sample is too thin to detectan antibody, so that the problem arises that the case of “true negative”cannot be differentiated from the case of “negative (false negative)”occasioned by the low concentration of the sample.

[0013] Furthermore, when samples lean in antibodies are to be tested, ahighly sensitive assay system is required but in that case there is theproblem that byproducts formed by nonspecific reactions due tocontaminants in the samples are liable to be simultaneously detected togive false positive results.

[0014] Therefore, an antibody assay system insuring sufficiently highdetection sensitivity even when such body fluids as urine and saliva areused as samples, that is to say a reliable assay system contributory toreduced chances for false negative and false positive tests because ofhigh specificity, is required.

[0015] The first object of the present invention is to provide anantibody assay technology (antibody assay method and antibody assaydevice) which is capable of detecting antibodies against sources ofinfection occurring in test samples such as body fluids with highsensitivity and high specificity.

[0016] The second object of the present invention is to provide anantibody assay method which enables determinations with high accuracythrough suppression of “false positive” reactions arising fromcontaminants in samples even when the samples are those of urine orother body fluid which are comparatively lean in the target antibody.

[0017] The third object of the present invention is to provide anantibody assay method as an improvement in immunocapillary assay orimmunochromatographic assay, by which the existence and amount of thetarget antibody as the object of detection in a sample can be accuratelydetermined with a clear demarcation between a “false negative reaction”arising from the nature of the sample and a “true negative” reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a diagram showing a solid phase support in the form of astrip as a constituent element of the antibody assay device of theinvention. In FIG. 1, the code 1 represents a first region, 2 a tracerregion, 3 a second region, 4 a third region, 5 a test zone and 6 acontrol zone.

[0019]FIG. 2 is a diagram illustrating the principle of assay of thetarget antibody in a sample with the antibody assay device of theinvention. The respective codes used have the same meanings as in FIG.1.

[0020]FIG. 3 is schematic diagrams showing a strip of solid phasesupport (A) and a housing (B) accommodating said solid phase supportincluded in the antibody assay device of the invention. In FIG. 3, thecodes 1˜6 have the same meanings as in FIG. 1, and the code 7 representsan upper section of the housing, 8 a lower section of the same, 9 asample inlet port, and 10 a detection window.

[0021]FIG. 4 is a diagrammatic representation of the results ofdetermination of anti -H. pylori antibody in urine in Example 1 (5) (i)In FIG. 4, the open circles represent data on urine samples fromsubjects with H. pylori infection who gave a positive ¹³C-UBT test andthe closed circles represent data on urine samples from subjects whogave a negative ¹³C-UBT test.

[0022]FIG. 5 is a diagrammatic representation of the data on anti-H.pylori antibody in urine as determined in Example 1 (5) (ii). In FIG. 5,the ordinate represents absorbance (O.D. 450 nm) and the abscissarepresents the H. pylori-positive and H. pylori-negative groupsestablished according to the ¹³C-UBT test.

[0023]FIG. 6 is a diagrammatic representation of the data on anti-H.pylori antibody in urine as determined in Example 1 (6). In FIG. 6, opencircles represent data on urine samples from subjects who gave apositive ¹³C-UBT test and closed circles represent data on urine samplesfrom subjects who gave a negative ¹³C-UBT test.

[0024]FIG. 7 is a diagrammatic representation of data on anti-HBcantibody in urine as generated in Example 2 (2). In FIG. 7, closedcircles represent data on urine samples from subjects who gave apositive test for blood anti-HBc antibody and open circles representdata on urine samples from subjects who gave a negative test for bloodanti-HBc antibody.

[0025]FIG. 8 is a diagram showing gel permeation chromatograms of urinesamples giving false positive reactions in the determination of anti-HIVantibody in urine and the antibody reactivity of each fraction (Example3 (1)). In FIG. 8, the ordinate represents absorbance (O.D.) and theabscissa represents the gel permeation chromatographic fraction(fraction No.). The solid line represents the absorbance of the proteinat 280 nm, the black dot-line represents data generated with anti-human(IgG+IgM) antibody, open triangle-line represents data generated withanti-human IgG (Fc-specific) antibody; and the closed triangle-linerepresents data generated with anti-human IgG (Fab-specific) antibody.

[0026]FIG. 9 is a diagrammatic representation of data on anti-H. pyloriantibody in urine as determined in Example 5. In FIG. 9, the ordinaterepresents absorbance (O.D. 450˜650 nm) and the abscissa represents theH. pylori-positive group (+: n=56) and −negative group (−:n=44) asclassified by the ¹³C-UBT test.

[0027]FIG. 10 is a diagrammatic representation of data on anti-rubellaantibody in urine as generated in Example 6. In FIG. 10, the ordinaterepresents absorbance (O.D. 450˜650 nm) and the abscissa represents theanti-rubella antibody-positive group (+:n=76) and −negative group(−:n=23) as classified according to the serum level measured with acommercial kit.

[0028]FIG. 11 is a diagram showing the test site and control site in thesecond region of the antibody assay device of the invention [Example 7(3)].

[0029]FIG. 12 is a histogram showing assay data on anti-H. pyloriantibody in the urine, whole blood and plasma as generated with theantibody assay device of the invention in comparison with thecorresponding data generated with control devices (A˜E). In FIG. 12,“Specificity” represents the percentage of negative tests (negativerate) relative to the total number of tests when samples from subjectsverified by the ¹³C-UBT test to be negative were determined for eachtest item with each assay device and “Sensitivity” represents thepercentage of positive tests (positive rate) relative to the totalnumber of tests when samples from subjects verified by the ¹³C-UBT testto be positive were determined for each test item with each assaydevice. The control devices A˜H mean the following devices.

[0030] A: Helitest (manufactured by Cortecs Diagnostics)

[0031] B: H. pylori-Check-1

[0032] (Manufactured by Bio-Medical Products)

[0033] C: First Check H. pylori

[0034] (Manufactured by Worldwide Medical Corp)

[0035] D: Biocard Helicobacter pylori IgG

[0036] (Manufactured by Anti Biotech Oy)

[0037] E: Insta Test H. Pylori

[0038] (Manufactured by Cortez Diagnostics Inc.)

[0039] F: One Step H. pylori Test

[0040] (Manufactured by Teco Diagnostics)

[0041] G: H. pylori SPOT

[0042] (Manufactured by International Immuno-Diagnostics)

[0043] H: Quick Stripe H. pylori

[0044] (Manufactured by Diatech Diagnostics Inc.)

DISCLOSURE OF INVENTION

[0045] The inventors of the present invention did much research forestablishing an assay system which would enable high-precisiondetermination of target antibodies even when samples are lean in theantibodies, for example urine samples, and found that an antibodycomponent which nonspecifically binds the antigen in an antigen-antibodyreaction (hereinafter referred to as the nonspecific binding antibodycomponent) exists in the assay system to give rise to nonspecificreactions, thus causing a false positive result and hence lowering theaccuracy of detection.

[0046] Based on the above findings the inventors did further researchand found that said nonspecific reactions can be suppressed byconducting the antigen-antibody reaction between the target antibody tobe assayed and the antigen specific to the particular antibody in thepresence of an Escherichia coli (E. coli) component, whereby the falsepositive rate can be reduced to achieve a significant improvement in theaccuracy of detection.

[0047] The inventors further discovered that said nonspecific bindingantibody component comprises IgG fragments and/or their denaturationproducts which retain the antigenicity of the light (L) chain or F (ab)region of the IgG and that this antibody component cross-reacts with theordinary antibody assay reagents (e.g. secondary antibodies) used inserum antibody assay systems, thus leading to false positive tests.

[0048] Based on the above findings, the inventors of the presentinvention further confirmed that the nonspecific reactions in anantibody assay system can be inhibited by using a reagent having aspecific affinity for the Fc region of the assay target antibody IgG asan antibody assay reagent, whereby the false positive rate can bereduced to improve the accuracy of detection in a significant degree.

[0049] Meanwhile, the inventors endeavored to improve the antibody assayhardware (the immunocapillary assay device and immunochromatographicassay device) and found that “true negative” reactions can be accuratelydetected excluding “false negative” reactions by establishing a “controlsite” for detecting an arbitrary antibody in samples in addition to thesite (test site) for detecting the target antibody in the reaction zone(evaluation zone) of the strip as a part of the assay device. Thus, insuch an assay system, when the sample is an inappropriate sample whichcannot be assayed for reasons such as too low a concentration of theantibody (that is to say the total amount of the antibody is too small),the “control site” gives a negative signal indicating that the sample isnot assayable. On the other hand, when the sample has an appropriateantibody concentration, the “control site” gives a positive signalindicating that the sample is appropriate for the intended assay of thetarget antibody. Then, according to the result in this “test site”, onemay know for certain the presence or absence of the target antibody inthe sample, that is to say whether the sample is “positive” or “truenegative”.

[0050] In this connection, Japanese Unexamined Patent Publication NO.299464/1989 and Japanese Unexamined Patent Publication NO. 167497/1994,both disclosing improvements in the antibody assay hardware(immunocapillary assay device and immunochromatographic assay device),describe the devices including a control site in addition to a testsite. However, the control site in these devices is designed toascertain whether or not a label disposed in an upstream region of thestrip has traversed through the test site by capillary action and,therefore, is quite different from the control site in accordance withthe invention.

[0051] The present inventors further confirmed that when the couplingreaction between the target antibody and the corresponding antigen bymeans of the above improved antibody assay device is conducted in thepresence of an E. coli component, the nonspecific reaction in thisantigen-antibody reaction system is inhibited and that when a reagenthaving a specific affinity for the Fc region of the IgG is used as theantibody assay reagent, the nonspecific reaction with theantigen-antibody complex is inhibited, thus leading to a significantdecrease in the incidence of a false-positive test.

[0052] The present invention has been developed on the basis of theabove several findings.

[0053] In a first aspect thereof, the present invention provides ahigh-precision method for assaying an antibody with a reduced incidenceof false positive reaction.

[0054] (1-1) As one mode thereof, the above antibody assay method fordetecting a target antibody in a sample by utilizing an antigen-antibodyreaction is characterized in that said reaction is carried out betweensaid antibody and an assay antigen in the presence of an E. colicomponent.

[0055] This method for assaying an antibody includes the followingspecific methods.

[0056] (a) The antibody assay method in which said E. coli component isat least one member selected from the group consisting of the solublefraction and lipopolysaccharide fraction of Escherichia coli.

[0057] (b) The antibody assay method wherein the E. coli component isused in a proportion of about 0.1˜100 μg, preferably about 0.5˜50 μg,per μg of the assay antigen.

[0058] (1-2) As another mode, the antibody assay method comprisesdetecting a target antibody in a sample by the sandwich technique,characterized in that a reagent comprising a secondary antibody having aspecific affinity for the Fc region of the target antibody IgG is usedas an antibody assay reagent.

[0059] This method for assaying an antibody includes the followingspecific methods.

[0060] (a) The antibody assay method in which the secondary antibody isan Fc-specific anti-IgG antibody.

[0061] (b) The antibody assay method comprising an antigen-antibodyreaction step in which the target antibody in the sample is coupled toan immobilized antigen specific to said antibody as immobilized on asupport and a reaction step in which the target antibody captured bysaid immobilized antigen is reacted with a secondary antibody having aspecific affinity for the Fc region of the antibody IgG.

[0062] (C) The above antibody assay method in which the antigen-antibodyreaction is carried out in the presence of an E. coli component.

[0063] In a second aspect, the present invention relates to an antibodyassay device. This device includes the following embodiments.

[0064] (2-1) An antibody assay device comprising a solid phase supporthaving at least (a) a first region to which a sample is applied and (b)a second region in which the antibody in the test sample is reacted asarranged in such a sequence that the sample is transported from thefirst region to the second region by capillary action, and a labelingmeans for detecting the result of reaction in the second region, said(b) second region having (i) a test site where a ligand for capturingthe target antibody to be detected has been immobilized and (ii) acontrol site where a ligand for capturing an arbitrary antibody in thesample has been immobilized.

[0065] (2-2) The antibody assay device wherein the ligand immobilized inthe test site is an antigen to the target antibody occurring in thesample.

[0066] (2-3) The antibody assay device wherein the ligand immobilized inthe control site is an anti-human immunoglobulin antibody capable ofcapturing an arbitrary antibody in the sample.

[0067] (2-4) The antibody assay device comprising a labeled ligand to bebound by both the target antibody and arbitrary antibody as saidlabeling means.

[0068] (2-5) The antibody assay device wherein the labeling means is alabeled ligand to be bound by both the target antibody and arbitraryantibody as removably supported upstreams of the second region of thesolid phase support in such a manner that, upon contact with a sample,it reacts with the target antibody and arbitrary antibody to form atarget antibody/labeled ligand complex and an arbitrary antibody/labeledligand complex, respectively, which are then transported by capillaryaction to the second region where they are fixed in the test site andcontrol site, respectively.

[0069] (2-6) The antibody assay device wherein the labeled ligand issupported in a region (tracer region) intermediate between the firstregion and second region of the solid phase support.

[0070] (2-7) The antibody assay device wherein the labeled ligand to bebound by both the target antibody and arbitrary antibody is a labeledanti-human immonoglobulin antibody.

[0071] (2-8) The antibody assay device wherein the anti-humanimmunoglobulin antibody is an anti-IgG antibody having a specificaffinity for the Fc region of immunoglobulin G.

[0072] (2-9) The antibody assay device wherein the solid phase supportis further provided with an absorption region downstreams of the firstand second regions so that the sample transported from the first regionto the second region is further transported by capillary action to theabsorption region.

[0073] (2-10) The antibody assay device wherein the coupling reaction ofthe target antibody at the test site in the second region takes place inthe presence of an E. coli component.

[0074] In a third aspect, the present invention relates to a method forsolid phase assay of a target antibody in a sample. This method includesthe following embodiments.

[0075] (3-1) A method for solid phase assay of a target antibody whichcomprises applying the sample to the first region of the antibody assaydevice and detecting the development of a color at the test site in thesecond region under the condition of the control site in the secondregion developing a color.

[0076] (3-3) The method for solid phase assay of a target antibodywherein the coupling reaction of the target antibody at the test site inthe second region of the antibody assay device takes place in thepresence of an E. coli component.

[0077] In a fourth aspect, the present invention relates to an antibodyassay reagent kit for use in association with said antibody assaydevice. The antibody assay reagent kit may include the followingembodiments.

[0078] (4-1) An antibody assay reagent kit characterized by itscomprising an E. coli component.

[0079] (4-2) The antibody assay reagent kit further comprising anantigen or antibody assay reagent which is optionally immobilized.

[0080] (4-3) The antibody assay reagent kit characterized by itscontaining an Fc-specific anti-IgG antibody as the antibody assayreagent.

[0081] (4-4) The antibody assay reagent kit containing the antibodyassay device according to the invention.

[0082] (1) Antibody Assay Method

[0083] In the first place, the antibody assay method as the first aspectof the present invention is now described in detail.

[0084] The antibody assay method of the invention represents animprovement in the antibody immunoassay method and is characterized inthat the incidence of false positive reaction can be decreased throughinhibition of non-specific reaction.

[0085] (1-1) As an embodiment of the above antibody assay method, therecan be mentioned a method in which the antigen-antibody reaction betweenthe target antibody in a sample and an antigen specific to said antibodyis carried out in the presence of an E. coli component. In accordancewith this method, the nonspecific reaction in the antigen-antibodyreaction is significantly inhibited, with the result that the incidenceof a false positive test can be decreased.

[0086] The E. coli component is not particularly restricted providedthat it is a component of Escherichia coli, thus including but notlimited to the protein component, carbohydrate component or lipidcomponent thereof or a mixture of such components. As a preferredexample, a soluble fraction or lipopolysaccharide (LPS) fraction of E.coli can be mentioned.

[0087] There is no particular limitation on the method for preparingsuch an E. coli component but a variety of methods can be selectivelyused. A usual procedure may comprise growing an arbitrary E. coli strainin a medium suited for its proliferation, harvesting the grown cells,and either disintegrating the cells physically by means of a sonicatoror solubilizing them with a surfactant or the like to provide a solublefraction (extract). The LPS mentioned above can be prepared by anextractive procedure using an organic solvent, e.g. phenol, chloroformor ether, or a mixture of two or three different organic solvents. Itcan also be prepared artificially using a genetic engineering technique.Moreover, commercial products can be expediently utilized (e.g.Lipopolysaccharide E. coli. which is available from Difco or Sigma)

[0088] The preferred sample to which the invention can be applied is abody fluid sample. The body fluid is not restricted provided that it isa body fluid derived from a human or other animal in which the targetantigen is supposedly contained. Thus, the term “body fluid” covers abroad variety of biological fluids which are used as samples in routinelaboratory tests. More particularly, the body fluid includes blood,inclusive of serum and plasma, urine, cerebrospinal fluid, amnioticfluid, saliva, sweat, and so forth. Particularly the present inventionsolves the problem of poor detection accuracy associated withnoninvasive samples which are favored as samples for antibody detection,such as urine, saliva and sweat, particularly urine, and, therefore,those biological materials can be mentioned as preferred examples of thebody fluid.

[0089] The “target antibody”, the object of determination, is notparticularly restricted provided that it is an antibody the detection ofwhich is desired, thus including antibodies against various sources ofinfection which are foreign bodies to the host.

[0090] The sources of infection are not particularly restricted butinclude many different pathogens which infect man and other animals andgive rise to antibodies in the hosts. More particularly, said pathogenincludes a variety of viruses such as HIV (human immunodeficiencyvirus), type A, B, C and other hepatitis viruses, rubella virus,influenza virus, measles virus, cytomegalovirus, herpes simplex virus,varicella-zoster herpes virus, adenovirus, enterovirus, etc.; bacteriasuch as Helicobacter pylori (hereinafter referred to briefly as H.pylori), Clamydia spp., Mycobacterium tuberculosis, spirochetes,gonococci, Treponema pallidum, Mycoplasma spp., etc. (excludingEscherichia coli); and protozoae such as Toxoplasma gondii, Entamoebahistolytica, Rickettsia tsutsugamushi, and so forth. Preferred areviruses such as HIV, hepatitis viruses, rubella virus, influenza virus,measles virus, herpes virus, etc. and bacteria represented byHelicobacter pylori etc., with bacteria such as H. pylori beingparticularly preferred.

[0091] The antigen for use in the antibody assay method of the inventionis not particularly restricted provided that it is an antigen capable ofundergoing antigen-antibody reaction with the target antibody to bedetected. Thus, for example, any of the antigens used in theconventional serum antibody assay system can be successfully used. Thoseantigens may not only be the very pathogens such as said viruses andbacteria but also be antigens having the antigenic determinant groupsintrinsic to the respective pathogens. Thus, for example, inactivatedpathogens available upon heat treatment or irradiation of pathogens,antigens prepared by extracting pathogens with a surfactant or the like,and antigens artificially prepared by chemical synthesis or recombinantDNA technology.

[0092] Incidentally, whether a candidate antigen may be successfullyused or not in the assay method of he invention can be easilyascertained typically by testing its reactivity with the target antibodyin the conventional manner.

[0093] In the assay method of the invention, said antigen may beoptionally used as immobilized on an arbitrary solid phase beforehand.The solid phase mentioned just above may be any of the various solidphases in routine use in this field of art, thus including but notlimited to sticks, beads, plates (inclusive of microtiter plates) andtest tubes made of various materials, for example glass, cellulosepowder, Sephadex, Sepharose, polystyrene, filter paper,carboxymethylcellulose, ion exchange resins, dextran, plastic film,plastic tubing, nylon, glass beads, silk, polyamine-methyl vinylether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acidcopolymer, etc.

[0094] The method for immobilization is not particularly restricted,either, but may be whichever of physical bonding and chemical bonding.For example, chemical bonding methods such as covalent bonding methods,e.g. diazo method, peptide method (acid amide derivative method,carboxyl chloride resin method, carbodiimide resin method, maleicanhydride derivative method, isocyanate derivative method, bromocyanactivated polysaccharide method, cellulose carbonate derivative method,condensing reagent method, etc.), alkylation method, crosslinking agentcoupling method (the method for coupling to a support usingglutaraldehyde, hexamethylene isocyanate or the like as the crosslinkingagent), Ugi reaction coupling method, etc.; ionic binding methods usingion exchange resins and the like supports; and physical adsorptionmethods using glass beads or other porous glass supports.

[0095] The amount of the antigen to be used in the assay system is notparticularly restricted but may be freely selected according to theamount of the antigen which is in routine use for the particular assaysystem. For example, when the sandwich method is used, generally theantigen is used in excess over the target antibody. Taking the case inwhich the reaction is conducted in a 100 μl reaction system as anexample, the antigen may be used in a proportion of generally about0.1˜100 μg/ml, preferably about 1˜10 μg/ml.

[0096] The conditions of the antigen-antibody reaction between saidantigen and target antibody are not particularly restricted but may bethe same as those in routine use for conventional immunoassays exceptthat the reaction should be conducted in the presence of an E. colicomponent. A typical procedure may comprise incubating or allowing tostand said antigen, antibody and E. coli component together at atemperature of generally not higher than 45° C., preferably about 4˜40°C., more preferably about 25˜40° C., for about 0.5˜40 hours, preferablyabout 1˜20 hours. The solvent for use in the reaction and its pH are notparticularly restricted, either, as far as the reaction is notinterfered with. Thus, the conventional buffers showing a buffer actionin the pH range of about 5˜9, such as citrate buffer, phosphate buffer,tris buffer, acetate buffer, etc. can be used generally in the routinemanner.

[0097] The proportion of the E. coli component in this reaction systemis not particularly restricted but may for example be generally about0.1˜100 μg, preferably about 0.5˜50 μg, per μg of the antigen in thereaction system.

[0098] The procedure for practicing the antibody assay method of theinvention is not particularly restricted except for the basicrequirement that it comprises an antigen-antibody reaction step in whichthe target antibody is reacted with the corresponding antigen, which maybe an immobilized antigen, in the presence of said E.coli component.Preferably, however, the method further comprises a step of detectingthe target antibody captured by said antigen (antigen-antibody complex),that is to say a step of reacting the antigen-antibody complex with anantibody assay reagent.

[0099] The method of detecting and quantitating the antigen-antibodycomplex obtained by said antigen-antibody reaction and the conditionsthereof are not particularly restricted but may be those in routine usefor immunoassays in general.

[0100] Preferably the present invention can be carried into practice bythe sandwich method. In the solid phase sandwich method, for instance,the target antibody in a sample can be assayed typically by thefollowing procedure.

[0101] First, an E. coli component and a sample supposedly containingthe target antigen (a body fluid such as urine) are added to a solidphase antigen which is an immobilized antigen capable of undergoing aspecific antigen-antibody reaction with the target antibody to therebycarry out an antigen-antibody reaction. After the unbound substances notcoupled to the solid phase antigen are removed by washing, for instance,an antibody assay reagent is added for reaction with the target antibodycoupled to the solid phase antigen (antigen-antibody complex) and theantigen-antibody complex is detected or quantitated by a detection meanscorresponding to the particular assay reagent.

[0102] The selection and modification of various means for such assaysare well known to those skilled in the art and any of such techniquescan be utilized in the practice of the present invention [e.g. “RinshoKensa-hou Teiyo (Outline of Clinical Test)”, Kanehara Publishing Co.,1995].

[0103] The antibody assay reagent for use here is not particularlyrestricted but includes a variety of reagents in routine use in the art.For example, secondary antibodies such as an anti-human immunoglobulinantibody capable of binding the objective antibody (immunoglobulin) canbe mentioned. The anti-human immunoglobulin antibody mentioned aboveincludes the antisera, purified products thereof (polyclonal antibodies)and monoclonal antibodies available from arbitrary animals immunizedusing an immunoglobulin in the class corresponding to the targetimmunoglobulin as an immunogen.

[0104] Further, as the antibody assay reagent, an anti-IgG antibodyhaving a specific affinity for the Fc region of the target antibody(IgG) can also be used. As such an anti-IgG antibody, an Fc-specificanti-IgG antibody which is not reactive to the light chain of IgG or theF(ab) region of IgG or protein A, protein G or the like which isspecifically reactive to the Fc region of IgG can also be used. Thesecan be used with particular advantage when the target antibody is anIgG.

[0105] Those antibody assay reagents can be prepared in the conventionalmanner or purchased from commercial sources.

[0106] For detection, the antibody assay reagent may be directlymodified with a conventional labeling agent or indirectly modified by anadditional detection means.

[0107] The labeling agent is not particularly restricted but any of theagents hitherto-known or expected to come into use in future can beemployed. To mention specific examples, radioisotopes such as ¹²⁵I, ³H,¹⁴C, etc.; enzymes such as alkaline phosphatase (ALP), peroxidase (e.g.HRP), etc.; fluorescent substances such as fluorescein isothiocyanate(FITC), tetramethylrhodamine isothiocyanate (RITC), etc.;1N-(2,2,6,6-tetramethyl-1-oxyl-4-piperidyl)-5N-(aspartate)-2,4-dinitrobenzene(TOPA), etc. can be used. The immunoassay methods using theabove-mentioned labeling agents are called radioimmunoassay, enzymeimmunoassay, fluoroimmunoassay, and spin immunoassay, respectively. Theimmunochromatoassay method using an antibody assay reagent prepared bylabeling colloidal gold-stained latex particles can also be employed.

[0108] Labeling with those labeling agents, modifications by indirectlabeling, and their detection can be made by the per se known methods[Tatsuo Iwasaki et al.: Monoclonal Antibody, Kodansha Scientific, 1984;and Eiji Ishikawa et al.: Enzyme Immunoassay, 2nd Edition, Igaku Shoin,1982, among others].

[0109] In the assay method of the invention, it is essential that an E.coli component be included in the reaction system comprising the targetantibody to be assayed and the corresponding antigen and as far as thisrequirement is satisfied, the rest of the basal procedure is notparticularly restricted but may be the same as that used in conventionalimmunoassays or routinely in the art. Therefore, the conditions of thereaction between said antigen-antibody complex and said antibody assayreagent are not particularly restricted, either, but may be the same asthose used in immunoassays in general. The commonest procedure comprisesincubating or allowing to stand the assay system under the sameconditions as those described above for the antigen-antibody reaction,i.e. generally at a temperature not over 45° C., preferably about 4˜40°C., more preferably about 25˜40° C. and a pH level of about 5˜9 forabout 0.5˜40 hours, preferably about 1˜20 hours.

[0110] The presence or absence of the target antibody in a sample or itscontent is evaluated by measuring the label activity, which depends onthe kind of labeling agent used in the labeling of the antibody assayreagent (or the indirect label), in the routine manner or in terms ofthe antibody titer calculated from the measured value.

[0111] (1-2) As an alternative mode of the antibody assay method of thepresent invention, there can be mentioned an immunological method forassay of the target antibody in a sample which comprises using a reagenthaving a specific affinity for the Fc region of the target antibody IgGas the antibody assay reagent.

[0112] In accordance with this method, the reaction between thenonspecific binding antibody component and the antibody assay reagentcan be significantly suppressed so that the frequency of false positivetests can be decreased. Thus, this antibody assay method may be regardedas an improvement in the sandwich method for immunoassay of antibodies.

[0113] The antibody assay reagent of the invention is reactive to thetarget antibody (IgG) and, therefore, can be used for detection of theantibody and is characterized in that it is not reactive to the lightchain of the target antibody IgG or the F(ab) region of the target IgG,that is to say it has a specific affinity for the Fc region of thetarget IgG.

[0114] More particularly, said antibody assay reagent may for example bean Fc-specific anti-IgG antibody which can be prepared by using the Fcregion of the target antibody IgG as the immunogen, and includesantisera, purified products thereof (polyclonal antibodies) andmonoclonal antibodies which can be obtained from arbitrary animalsimmunized with said immunogen. This reagent is not limited to suchantibody preparations but may be protein A, protein G or the like whichis specifically reactive to the Fc region of the antibody IgG.

[0115] Those antibody assay reagents can be prepared in the routinemanner or purchased from commercial sources (e.g. Sigma, Cappel orJackson Immuno Research Laboratories,Inc.).

[0116] For detection, the antibody assay reagent may be directlymodified with a conventional labeling agent or indirectly modified by anadditional detection means.

[0117] The kind of labeling agent, method for labeling, and method ofdetecting the label can be the same as those mentioned under (1-1).

[0118] The antibody assay method of the invention comprises the use ofthe above-mentioned antibody assay reagent as an essential featurethereof in the detection of the target antibody, i.e. theantigen-antibody complex, and as far as this requirement is met, therest of the basal procedure may be liberally the same as that used inconventional immunoassays by the sandwich technique.

[0119] Basically, the antibody assay method of the present invention iscarried into practice by reacting an antigen capable of reacting withthe target antibody in a sample and detecting the target antibody boundto the antigen (antigen-antibody complex) with said antibody assayreagent.

[0120] The assay sample and the antigen may respectively the same asmentioned under (1-1), and as to the target antibody, too, the sameantibodies as those mentioned under (1-1) can be used, provided that theantibody is an antibody IgG against the infection source. In the methodof the present invention, the infection source may include Escherichiacoli.

[0121] Where necessary, the antigen or antibody assay reagent in thepresent invention can be used as immobilized on an arbitrary solidphase. The solid phase for use and the method of immobilization may forexample be the same as mentioned under (1-1).

[0122] The antigen-antibody reaction between the antigen and the targetantibody is not particularly restricted but can be conducted under theconditions in routine use for conventional immunoassays. A typicalprocedure comprises incubating or allowing to stand a reaction systemcomprising the antigen and target antibody generally at a temperature ofnot over 45° C., preferably about 4˜40° C., more preferably about 25˜40°C. for about 0.5˜40 hours, preferably about 1˜20 hours.

[0123] Although it is not mandatory, an E. coli component may be causedto be present in the antigen-antibody reaction as mentioned under (1-1).

[0124] The resulting antigen-antibody complex is then washed andsubmitted to a step in which it is reacted with said specified antibodyassay reagent. This reaction can be carried out under the sameconditions as are generally used in, or substantially in the same manneras, the conventional immunoassays (sandwich assays). The solvent, forinstance, is not particularly restricted provided that it does notinterfere with the reaction, thus including but not limited to buffersat pH about 5˜9, such as citrate buffer, phosphate buffer, tris bufferand acetate buffer, to mention just a few examples. The reaction timeand reaction temperature are not particularly restricted, either, butmay for example be the same as those mentioned for said antigen-antibodyreaction.

[0125] The presence or absence of the target antibody in a sample or itscontent is evaluated by measuring the label activity, which depends onthe kind of labeling agent used in the labeling of the antibody assayreagent (or the indirect label), in the routine manner or in terms ofthe antibody titer calculated from the measured value, just as mentionedunder (1-1).

[0126] (2) Antibody Assay Device

[0127] The antibody assay device according to the second aspect of theinvention is now described in detail.

[0128] The antibody assay device of the invention is an improved methodby which the presence and/or quantity of the target antibody to bedetected in a sample can be determined with good accuracy by a solidphase assay procedure.

[0129] More particularly, the antibody assay device of the invention isan assay device comprising a solid phase support having at least (a) afirst region for contact with a sample and (b) a second region forreaction of the antibody in the sample as arranged in such a sequencethat the sample is transported by capillary action from said firstregion to said second region, and a label means for detecting the resultof reaction in said second region, characterized in that said (b) secondregion has (i) a test site where a ligand for the target antibody to beassayed is immobilized and (ii) a control site where a ligand forcapturing an arbitrary antibody in the sample is immobilized.

[0130] The outstanding feature of the antibody assay device of theinvention is that a control site independent of a test site is providedin the second region, which control site is such that, when a propersample is applied and tested in a proper manner, it forms an indicationrepresenting a positive test in the presence of a label regardless ofwhether the target antibody is present or not in the sample while, whenan improper sample is applied or a sample is tested in an impropermanner, it forms an indication representing a negative test in thepresence of a label regardless of whether the target antibody is presentor not in the sample.

[0131] Thus, the control site in the second region of this device is asite giving an indication of whether the result (particularly thenegative result) in the test site is a valid assay result regardless ofthe presence or absence of the target antibody in the sample. With theantibody assay device of the invention, thanks to the aboveconstruction, it is possible to determine, qualitatively andquantitatively, the antibody in the sample with high accuracy (highreliability) with a clear distinction between false negative and truenegative results.

[0132] Furthermore, the antibody assay device of the invention may be sodesigned that the reaction of the target antibody in the test site iscarried out in the presence of an E. coli component or so designed thata reagent having a specific affinity for the Fc region of the targetantibody IgG is used as the antibody assay reagent for detecting theresult of reaction in the test site. With the antibody assay device ofthe invention, thanks to the above-described construction, nonspecificreactions are inhibited so that the incidence of a false positive testis significantly decreased, thus making it possible to determine,qualitatively and quantitatively, the target antibody in the sample withgood accuracy and high sensitivity.

[0133] As the assay sample and target antibody for this antibody assaydevice of the invention, those mentioned under (1) may for example beemployed.

[0134] The present invention first provides a solid phase supportcomprising at least a first region and a second region.

[0135] The first region is a zone where the sample applied comes intocontact with the device and the second region is a zone where theantibody (the total antibody which may contain the target antibody) inthe sample undergoes reaction and coupling in the ligand-receptor modeor in the antigen-antibody mode and the result of reaction is displayedin the presence of a label (reaction zone and evaluation zone). Thoseregions are arranged on a solid phase support in such-a manner that thesample applied and coming into the first region is transported bycapillary action from said first region to the second region.Preferably, said regions are so arranged that all or at least a portionof the sample coming into the first region travels, by capillary action,through a substantially planar layer of the solid phase support to thesecond region.

[0136] Optionally the solid phase support may have a third regiondownstreams of the second region, as a region which absorbs the sample(liquid) migrating, by capillary action, from the first region to thesecond region and further downstreams.

[0137] The preferred solid phase support is formed in the shape of astrip and said first and second regions are arranged on one and the sameplane of the strip in such a manner that the sample applied travels, bycapillary action, from a first band (first region) to a second band(second region) and optionally further to a third band (third region).While the preferred form of said solid phase support is a strip asmentioned above, any other shape or geometry can be employed as far asthe functions expected of the solid phase support in the presentinvention can be implemented.

[0138] The solid phase support is capable of absorbing the sample(liquid) and, when wetted with the sample, allowing at least theantibody in the sample to travel, by capillary action, from the firstregion to the second region of the solid phase support and optionallyfurther to the third region. Moreover, the solid phase support ispreferably one that is capable of supporting and immobilizing a ligandwhich reacts with the antibody (inclusive of the target antibody)contained in the sample to capture the latter.

[0139] The proper solid phase support includes a variety of porousmaterials, e.g. polyethylene, glass fiber, cellulose, rayon, nylon,crosslinked dextran, various types of chromatograph paper,nitrocellulose, and filter paper.

[0140] The first region, second region and third region, for instance,of the solid phase support may respectively be constituted by the sameor different members selected from among the above-mentioned materials,with the choice of materials depending on the roles and functions of therespective regions.

[0141] The first region of the solid phase support is preferablyconstituted by a porous material adapted to absorb the sample appliedonto its surface and let it travel, by capillary action, to the secondregion. The porous material suited for the first region is notparticularly restricted but generally polyethylene (for example POREX:Porex Technologies, Fairburn, Ga.), glass fiber, rayon, nylon, andcellulosic materials inclusive of paper can be used. The preferredmaterial is a porous polyethylene or a cellulosic material such asfilter paper.

[0142] The second region of the solid phase support is preferablyconstituted by a porous material which is capable of allowing the sample(liquid) to be wicked by capillary action from the first region to thesecond region and supporting a ligand for the antibody (inclusive of thetarget antibody) occurring in the sample in a condition not dislodged bycapillary action. The porous material having such properties includesfilter paper, chromatograph paper, glass fiber, crosslinked dextran,nylon, nitrocellulose, etc. The preferred material is nitrocellulosebecause the ligand can be easily immobilized thereon.

[0143] The second region is provided with a test site where a ligandadapted to specifically recognize the target antibody in the sample andcapture it has been immobilized and a control site where a ligandadapted to recognize an arbitrary antibody in the sample and capture ithas been immobilized. The control site is disposed away from the testsite with a given interval therebetween, preferably downstreams in thedirection of capillary flow, and it is preferably so arranged that bothsites are contacted by the sample liquid front under the identicalconditions.

[0144] The ligand for the test site is not particularly restrictedprovided that it will be specifically coupled to the target antibody tobe assayed but is preferably an antigen which is specifically recognizedand bound by the target antibody (antigen-antibody reaction). As theantigen mentioned just above, those antigens which are in routine use inthe conventional serum antibody assay system can be liberally used.Those antigens may be the very pathogens such as viruses and bacteriabut may also be substances containing antigenic determinant groupsintrinsic to the respective pathogens. Thus, for example, the pathogensinactivated by heating or irradiation, the antigens obtained byextracting the pathogens with a surfactant or the like, andthoseantigens which are artificially prepared by chemical synthesis orrecombinant DNA technology can be mentioned.

[0145] The ligand for the control site is not particularly restrictedprovided that it couples an arbitrary antibody in the sample but ispreferably an antibody (anti-immunoglobulin antibody) which specificallyrecognizes and binds an arbitrary antibody in urine.

[0146] Those ligands are immobilized on the porous material in the testsite and control site, respectively, so that they will not be dislodgedfrom the respective sites by the capillary flow of the liquid sample.Thus, each ligand is bound to the corresponding site on the poroussupport so that it will not be caused to diffuse when the second regionis wetted by the sample containing the target antibody but be retainedstationary in the site without being transported to the third region ofthe solid phase support.

[0147] Immobilization of the ligands on said porous support can beachieved by the techniques well known to those skilled in the art, i.e.by physical bonding or chemical bonding.

[0148] Thus, for example, chemical bonding methods such as covalentbonding methods, e.g. diazo method, peptide method (acid amidederivative method, carboxyl chloride resin method, carbodiimide resinmethod, maleic anhydride derivative method, isocyanate derivativemethod, bromocyan activated polysaccharide method, cellulose carbonatederivative method, condensing reagent method, etc.), alkylation method,crosslinking agent coupling method (the method for coupling to a supportusing glutaraldehyde, hexamethylene isocyanate or the like as thecrosslinking agent), Ugi reaction coupling method, etc.; ionic bindingmethods; and physical adsorption methods can be mentioned. Whennitrocellulose is used as the porous support for the second region, saidligand can be conveniently immobilized by non-covalent bonding.

[0149] The amount of the ligand (antigen) for use in the test site ofthe second region is preferably in excess so that essentially all of thetarget antibody presumably present in the sample will be bound to thetest site.

[0150] The amount of the ligand (anti-immunoglobulin antibody) for usein the control site of the second region is also preferably in excess sothat essentially all of the arbitrary antibody (which may contain thetarget antibody) in the sample will be bound to the control site.

[0151] The coupling reaction between the target antibody and the ligand(particularly an-antigen) in the test site is preferably carried out inthe presence of an E. coli component. The E. coli component may besupplied to the reaction system by incorporating it in a dilution of theassay sample and applying the mixture to the first region or may beremovably immobilized in the test site of the solid phase supportupstreams of the test site (e.g. said first region or the tracer regionto be described hereinafter). The E. coli component is not particularlyrestricted provided that it is derived from Escherichia coli asmentioned above. Thus, it may be the protein fraction, carbohydratefraction or lipid fraction of the cells or a mixture of such fractions.The soluble fraction obtained by extraction of the cells or thelipopolysaccharide (LPS) fraction can be mentioned as a preferredexample.

[0152] As the sample is applied to the first region, the samplemigrates, by capillary action, to the second region where the targetantibody in the sample is bound and fixed to the test site within thesecond region and, then, the remaining arbitrary antibody (inclusive ofthe residue of the target antibody which has not been bound to the testsite) is bound and fixed to the control site downstreams of the testsite.

[0153] The labeling means in the present invention is used to detectwhether the target antibody and arbitrary antibody in the sample havebeen coupled and fixed to said test site and control site, respectively.

[0154] The labeling means may consist of a ligand for the antibody and adetectable label coupled to said ligand.

[0155] The ligand for the antibody is not particularly restricted as faras it is a molecule which recognizes and binds the antibody present inthe sample but, in the present invention, is preferably one which bindsnot only the target antibody to be assayed but also the arbitraryantibody present in the sample. As an example of such ligand, there canbe mentioned the same ligand as that mentioned for the control site,particularly the same anti-immunoglobulin antibody as that adopted asthe ligand in the control site.

[0156] The anti-immunoglobulin antibody mentioned above includesantisera available from arbitrary animals immunized with a relevantiumunoglobulin, e.g. a human immunoglobulin, as the immunogen,purification products thereof (polyclonal antibodies) and monoclonalantibodies.

[0157] The anti-immunoglobulin antibody as the ligand in the controlsite may optionally be an antibody directed to all classes of antibodiesso that it may capture and detect the total antibody in the sample or bean antibody directed to a desired class of antibody such asimmunoglobulin G (IgG). Preferably, the ligand is an anti-immunoglobulinantibody directed to antibodies in the same class as the class to whichthe target antibody belongs and this arrangement is preferred in that asantibodies of the same class as the target antibody are thus detected inthe control site, the optimum indication is obtained there for judgingwhether the assay has been done properly not only when a urine sample isused but when other body fluids such as sera are used as samples.

[0158] When the target antibody is IgG, the ligand as said labelingmeans is more preferably a ligand characterized by having a specificaffinity for the Fc region of the antibody IgG and, as preferredexamples of such ligand, an Fc-specific anti-IgG antibody which is notreactive to the light chain of the antibody IgG or the F(ab) regionthereof or Protein A, Protein G or the like having a specific reactivityto the Fc region of the antibody IgG can be mentioned. Suchanti-immunoglobulin antibodies or ligands can be prepared in the routinemanner or purchased from commercial sources.

[0159] The detectable labeling component is not particularly restrictedas far as it is a detectable label which is known to be useful forspecific binding assays, particularly immunoassays, or which will bepossibly used in the future [Tatsuo Iwasaki et al: Monoclonal Antibody,Kodansha Scientific, 1984; Eiji Ishikawa et al: Enzyme Immunoassay, 2ndEdition, Igaku Shoin, 1982, etc.].

[0160] The preferred label is one which undergoes change in color in thetest site or control site of the second region. Though not restricted, alabel undergoing a change of color which can be visually recognizedwithout the aid of any instrument is particularly preferred. Forexample, various chromogens such as fluorescent substances and absorbingdyes can be mentioned. The still more preferred is a label in the formof a powder containing a visually detectable marker.

[0161] The suitable particulate label includes polymer particles (e. g.latex or polystyrene beads) , sacks, liposomes, metallic gels (e.g.colloidal silver, colloidal gold, etc.), and polystyrene dye particles.Among them, metal gels such as colloidal silver and colloidal gold arepreferred.

[0162] While such a label can be coupled to a ligand, either chemicallyor physically, in the conventional manner to provide a labeled ligand,commercial products can also be utilized.

[0163] The labeling means which can be used in the present invention maybe any label that, when applied to the second region (inclusive of thetest site and control site) of the solid phase support, indicates theresult of the reaction with the antibody occurring in the sample whichhas taken place in the second region and, as far as this function can beachieved, there is no particular limitation on the mode of its presence.For example, when the assay device of the present invention is providedin the form of a flow through device, the labeling means may be includedin the antibody assay reagent kit independently of the solid phasesupport.

[0164] Preferably, the labeling means is removably immobilized on asolid phase support, and more preferably it is removably immobilizedupstreams of the second region of the solid phase support. The stillmore preferred mode is such that the labeling means is supported in aregion (hereinafter referred to as tracer region) intermediate betweenthe first and second regions of the solid phase support. In this mode ofuse, the sample applied to the first region is wicked by capillaryaction to the tracer region where it comes into contact with the labeledligand to form the target antibody/labeled ligand complex and thearbitrary antibody/labeled ligand complex. After passage through thetracer region, the sample containing those complexes migrates, bycapillary action, further to the second region. The ligand (antigen)disposed in the test site within the second region is specific to thetarget antibody and the ligand (anti-immunoglobulin antibody) disposedin the control site is specific to the arbitrary antibody. Therefore,the target antibody/labeled ligand complex which has migrated bycapillary action is first captured in the test site and the remainingarbitrary antibody/labeled ligand-complex (inclusive of the targetantibody/labeled ligand complex) in the sample is then captured in thecontrol site.

[0165] The tracer region of the solid phase support is not particularlyrestricted as far as it is a support capable of transporting the testsample containing the target antibody and arbitrary antibody from thefirst region to the second region and supporting said labeled ligand insuch a manner that the latter may be released by the capillary flow.Generally, it is a porous member made of polyethylene, glass fiber,rayon, nylon or a cellulosic material inclusive of paper. The preferredis a material which hardly allows nonspecific adsorption, for exampleglass fiber optionally treated with polyvinyl alcohol (PVA).

[0166] Among preferred embodiments of the invention is an embodimentwherein said tracer region and said test site in the second region aredisposed with a given interval therebetween on a solid phase support. Assuch an interval region is provided between the tracer region and thetest site, the antibody in the sample coming into contact with thelabeled ligand in the tracer region is blended with the labeled ligandthere before the sample reaches the test site in the second region,whereby the coupling reaction between the antibody and the labeledligand is more positively assured. Thus, this region provides anincubation environment (time and space) for the coupling of the targetantibody and arbitrary antibody with the labeled ligands prior tocontact of the target antibody and arbitrary antibody in the sample withthe test site and control site, respectively.

[0167] Furthermore, the solid phase support of the present invention mayhave a third region downstreams of the second region.

[0168] The sample continues migrating from the first region to theoptional tracer region to the second region and further to the thirdregion by capillary action. Thus, the third region functions as a regionreceiving the liquid coming from the second region by capillary action.

[0169] Generally the third region need only discharge the function ofreceiving the liquid component not bound in the second region but may befurther provided with a site informative of the completion of an assayupon advance of the capillary flow of the sample (that is the liquidfront) to a predetermined end-point zone on the solid phase support.

[0170] For the above purpose, the solid phase support may be providedwith a visible indicator zone containing a water-soluble dye such aserythrosine B, saffranine O, phenol red or the like downstreams of thesecond region. In this case, as the liquid front of the sample traversesthe second region into the third region, it flows through said indicatorzone and the dye disposed in this zone is carried downstreams by thecapillary flow of the sample (liquid), thereby informing that the samplehas already passed serially through, the first region, tracer region andsecond region (test and control sites) and accordingly that the assayhas just been completed.

[0171] The material for the third region is not particularly restrictedas far as it is capable of absorbing the sample (liquid), thus includingporous films or sheets of polyethylene, rayon, nylon and cellulosicmaterials inclusive of paper. The preferred material is a cellulosicmaterial such as paper.

[0172] The present invention is now described in detail, reference beinghad to the accompanying drawings showing the antibody assay device andits constituent members. It should, however, be understood that theillustrated device is a mere embodiment of the invention and by no meansdefinitive of the invention.

[0173]FIG. 1 is a diagram showing a solid phase support in the form of astrip (60×5 mm) as a member of the device of the invention. In FIG. 1,the code 1 represents a first region (16×5 mm, 0.92 mm thick) where anassay sample is applied and brought into contact with the strip; thecode 2 represents a tracer region (5×5 mm, 0.79 mm thick) where alabeled ligand (e.g. colloidal gold-labeled human IgG antibody) isimmobilized; the code 3 represents a second region (18×5 mm, 0.1 mmthick) where the reaction with the antibody in the sample takes placeand the result is indicated; and the code 4 represents a third region(22×5 mm, 1.46 mm thick) where the sample which has migrated from thefirst region and second region is absorbed.

[0174] The thickness of the first region is generally 0.2˜2 mm andpreferably 0.8˜1.2 mm, and the thickness of the tracer region isgenerally 0.2˜1.5 mm and preferably 0.5˜1 mm. The thickness of thesecond region is generally 0.03˜0.2 mm and preferably 0.08˜0.12 mm, andthe thickness of the third region is generally 0.5˜3 mm and preferably1˜2 mm. However, these ranges are not critical.

[0175] Disposed within said second region (3) is a test site (test line,about 1×5 mm) (5) where a ligand having a specific affinity for thetarget antibody is supported in position and a control site (controlline, about 1×5 mm) (6) where a ligand having an affinity for thearbitrary antibody (anti-human immunoglobulin antibody) is supported inposition. The test site (5) is disposed at a given distance (about 6 mm)from the tracer region and the control site (6) is disposed at a givendistance (about 6 mm) from the test site (5). The test site has thefunction of reacting specifically with the target antibody in the sampleand indicating in the presence of a label whether the target antibodyexists or not in the sample and the control site has the function ofindicating in the presence of a label whether the sample applied isproper or not.

[0176] The materials (supports) for the first region, tracer region,second region and third region constituting the solid phase supportstrip may have been simply connected in series in the longitudinaldirection of the strip along which the sample migrates and there is norestriction to the mode of connection. Preferably, however, theconnection between the longitudinally front end of the first region andthe rear end of the tracer region, the connection between the front endof the tracer region and the rear end of the second region, and theconnection between the front end of the second region and the rear endof the third region are respectively in superimposed relation. Morepreferably, as illustrated in FIG. 1, the longitudinally front end partof the first region is superimposed on the rear end part of the tracerregion and the longitudinally front end part of the tracer region issuperimposed on the rear end part of the second region. The rear endpart of the third region may be superimposed on the front end part ofthe second region. In such a mode of connection, the sample applied tothe first region is allowed to migrate smoothly in the longitudinaldirection of the strip. The width of the strip in its longitudinaldirection is not particularly restricted but may for example be 0.5˜10mm, preferably 1˜2 mm, more preferably 0.8˜1.2 mm. It should beunderstood that the top/bottom relationships of the respective regionsin the superimposed strip structure are not limited to those mentionedabove but may be reversed.

[0177] For convenience in use, the above solid phase support ispreferably packaged in the form of an assay unit.

[0178] The principle of assay with the assay device of the invention isnow explained with reference to FIG. 2.

[0179] (3) Solid Phase Assay Method

[0180] The third aspect of the invention is directed to a solid phaseassay method using the above-described device, which specifically is asolid phase method for assay of the target antibody in a sample whichcomprises bringing the sample into contact with the first region of theantibody assay device and detecting the development of a color in thetest site of the second region under the condition in which the controlsite of the second region is indicating a color.

[0181] In this assay, a sample suspected to contain the target antibodyis first applied to the first region (1) of the solid phase support.

[0182] For application of the sample, a body fluid such as urine may beused as it is or after dilution with a suitable diluent. The diluent isnot particularly restricted but includes various buffers having a bufferaction within the range of pH about 5˜9, preferably about 6.5˜8.5, (e.g.citrate buffer, phosphate buffer, tris buffer, acetate buffer, boratebuffer, etc.), surfactants, etc.

[0183] Since nonspecific reactions in the antigen-antibody reaction canbe suppressed to reduce the incidence of a false positive test byconducting the antigen-antibody reaction in the presence of an E. colicomponent, it is preferable to use a diluent containing such an E. colicomponent. The amount of the E. coli component (LPS) to be incorporatedin the diluent is not particularly restricted but is preferably suchthat about 0.1˜10 μg, preferably about 0.5˜5 μg, of said component willbe available per μg of the ligand (antigen) in the reaction system, i.e.the test site.

[0184] The amount of the E. coli component (LPS) to be incorporated inthe sample may for example be generally not less than 5 μg/ml,preferably 5˜100 μg/ml, more preferably 10˜50 μg/ml. Although the use ofan amount over 100 μg/ml is not prohibitive, the effect of the inventioncan be accomplished at the addition level of up to 100 μg/ml.

[0185] As the sample is applied to the first region (1), the firstregion 1 is wetted. The sample applied flows through the first region(1) into the tracer region (2) by capillary action and comes intocontact and reacts with the labeled ligand (colloidal gold-labeledanti-human IgG antibody) removably supported in the tracer region.

[0186] When a suitable sample is used and the target antibody iscontained in the sample, both the target antibody and arbitrary antibodycontained in the sample are coupled to said labeled ligand in the tracerregion (2) to form a target antibody/labeled ligand complex and anarbitrary antibody/labeled ligand complex, respectively. After passageof the same through the tracer region (2), the respective complexes orthe labeled ligand not forming such complexes are transported togetherwith the sample downstreams of the tracer region (2). In a preferredmode, the labeled ligand not forming a complex yet is given a sufficienttime (space) for forming complexes as it travels with theantibody-containing sample from the tracer region (2) to the test site(5) of the second region (3) by capillary action. As the sample reachesthe test site (5) in the second region, the target antibody/labeledligand complex in the sample is coupled to the ligand supported in thetest site (5) and immobilized in situ. The sample further migratesdownstreams by capillary action to reach the control site (6) where thearbitrary antibody/labeled ligand complex is coupled to the ligand(anti-human immunoglobulin antibody) in that site and immobilized there.Then, by detecting the complexes immobilized in the test site (5) andcontrol site (6) in the second region according to the label componentof the labeled ligand, the assay result can be indicated as a positivetest. In contrast, when a sample not containing the target antibody isapplied, said target antibody/labeled ligand complex to be immobilizedin the test site (5) is not formed so that the label is not detected inthis test site (5) (a negative test).

[0187] In this connection, the negative test indication in the test site(5) includes both a negative (false negative) test due to a lowconcentration of the sample (i.e. a small total amount of antibody inthe sample) and a negative (true negative) test due to the absence ofthe target antibody in the sample. These negative tests cannot bedifferentiated from each other according to the result in the test site(5) alone. However, in the case of a false negative test, said arbitraryantibody/labeled ligand complex to be immobilized in the control site(6) is not formed so that the control site gives a negative indication,while in the case of a true negative test, said arbitraryantibody/labeled ligand complex is formed so that the control site givesa positive indication. Therefore, according to whether the indication atthe control site is negative or positive, it is possible to tell whetherthe negative result in the test site (5) is a false negative test or atrue negative test. Furthermore, when a labeled ligand which has beendeactivated is used or otherwise a proper assay is not performed in aproper system, the control site (6) gives a negative indication so thatthe finding of a false negative test owing to such causes can beprevented.

[0188] The sample liquid containing all the unbound antibodies, labeledligands, etc. continues to migrate further downstreams of the secondregion (3) to the third region (4). Optionally an indicator zone may beprovided and, in this case, the liquid front coveys the dye from theindicator zone to the end-point zone, thus indicating the passage of theliquid and dye through the third region and completion of the assay.

[0189]FIG. 3 shows an example of the antibody assay device of theinvention for use in horizontal position. The solid phase support (A)comprising the first region (1), tracer region (2), second region (3)(including the test site (5) and control site (6)) and third region (4)is accommodated in a housing (B) made of a suitable material. Thehousing material is preferably a moldable plastic material such aspolystyrene, although other materials such as glass, metal and paper canalso be used. The housing consists of an upper section (7) havingseveral apertures and a lower section (8), and the solid phase supportis disposed on the lower section of the housing and covered with theupper section on top thereof. The apertures (9) and (10) in the uppersection of the housing are disposed in alignment with the series ofregions of the solid phase support and in the positions corresponding tothe first region (1) and second region (3), respectively, of the solidphase support.

[0190] The sample can be applied to the first region (1) of the supportfrom the aperture (9) (sample feeding port). The aperture (9) ispreferably provided with a projecting peripheral wall around it so thatthe wall may assist in the dripping of the liquid sample onto the firstregion of the support. The method for contacting the sample with thefirst region of the solid phase support is not particularly restrictedbut the sample is preferably dripped from said sample feeding portperpendicularly to the plane of the solid phase support.

[0191] The aperture (10) is disposed in a position permitting a visualaccess to the test and control sites in the second region of the solidphase support, whereby the fixation of the labeled ligand/targetantibody complex in the test site and that of the labeledligand/arbitrary antibody complex in the control site can be visuallyconfirmed (detection port, evaluation port). The aperture (10) need notnecessarily be a single port permitting a visual access to both the testsite and control site but may comprise two independent ports for thetest site and the control site, respectively.

[0192] With the antibody assay device of the present invention, thepresence or absence of the target antibody in a sample as well as theamount of the antibody can be determined with high accuracy by leavingthe assay system standing for a few minutes to 30 minutes, preferably5˜20 minutes, after application of the sample, generally at atemperature of not over 45° C., preferably 4˜40° C., more preferablyabout 15˜30° C.

[0193] (4) Antibody Assay Reagent Kit

[0194] The antibody assay method described under (1) or the slid-phaseassay method using the antibody assay device described under (2) can bemore expediently carried out when an antibody assay reagent kitcontaining a complete set of various reagents and equipment necessaryfor determination of the antibody is utilized.

[0195] The present invention, thus, further provides an antibody assayreagent kit for reducing to practice said antibody assay method and saidsolid phase assay method.

[0196] The antibody assay reagent kit according to the present inventionis intended for use for the purpose of detecting and quantitating anantibody in a sample through an antigen-antibody reaction and, as onepreferred mode, includes a kit containing said E. coli component as akit component. This reagent kit further contains an optionallyimmobilized antigen adapted to undergo antigen-antibody reaction with atarget antibody to be assayed, an antibody assay reagent, and so forth.Furthermore, for convenience in assaying, this reagent kit may furtherinclude a suitable reaction medium, diluent, wash buffer, reactionstopper and/or label activity test reagent.

[0197] Moreover, as another mode, the antibody assay reagent kit of theinvention may include a reagent having a specific affinity for the Fcregion of the target antibody IgG, preferably an Fc-specific anti-IgGantibody.

[0198] As a further alternative mode, the antibody assay reagent kit ofthe invention may contain said antibody assay device. The reagent kitmay further contain said E. coli component or said substance having aspecific affinity for the Fc region of the target antibody IgG or bothas reagents. Moreover, the kit may further contain such accessories as apipette and an ampoule (tube) for use in dilution of the sample inaddition to said reaction medium, diluent, wash buffer, reactionstopper, and label activity test reagent, among other reagents.

BEST MODE FOR CARRYING OUT THE INVENTION

[0199] The following examples are intended to illustrate the presentinvention in further detail and should by no means be construed asdefining the technical scope of the invention. It should be understoodthat many changes and modifications may be made by those skilled in theart easily on the basis of the foregoing disclosure of the inventionwithout departing from the technical scope of the invention.

EXAMPLE 1 Assay of H. pylori

[0200] (1) Preparation of H. pylori Antigen

[0201]H. pylori (a clinical isolate) was cultured on Brucella agarmedium (Becton) for 48 hours (10% CO₂, 5% O₂, 37° C.) and the growncells were harvested into cold PBS. The cells were centrifugally washedwith cold PBS for a total of 5 times, and cold PBS was added so as tomake a cell concentration of 100 mg/ml. Under stirring, one equivalentof cold 0.2% Triton X-100/PBS was added. The mixture was stirred for 5minutes and centrifuged and the supernatant was recovered as H. pyloriantigen solution and stored at −80° C.

[0202] (2) Preparation of an H. pylori Antigen Plate

[0203] The above H. pylori antigen solution (2.5 μg protein/ml) wasadded to a 96-well plate, 100 μl/well, and incubated at 4° C. overnight.After the wells were washed with PBS once, a blocking solution(Dulbecco-PBS [D-PBS], 1% BSA, 5% sorbitol, 0.05% NaN₃ [pH 7.4]) wasadded, 300 μl/well, and the plate was incubated at 4° C. overnight.After the blocking solution was discarded, the plate was dried at 25° C.overnight, sealed, together with a desiccant, in an aluminum bag andstored at 4° C. until used.

[0204] (3) Preparation of an E. coli Component

[0205]Escherichia coli (pvc18/JM109, Takara Shuzo) was cultured inampicillin-containing liquid LB medium (Luria-Bertani medium, NipponSeiyaku) at 37° C. for 18 hours. The culture was centrifuged to harvestthe cells, which were washed with 2 portions of PBS. To the washed cellswas added cold PBS to make 100 mg/ml, and the mixture was disrupted andextracted using a sonicator (10 seconds×3). The supernatant wasrecovered for use as an E. coli component and stored at −80° C.(hereinafter referred to as E. coli extract).

[0206] (4) Determination of anti-H. pylori Antibody in Urine

[0207] Using urine as the sample, the anti-H. pylori antibody in thesample was determined.

[0208] To each well of the H. pylori antigen plate prepared under (2),25 μl of a first buffer solution (200 mM Tris-HCl buffer, 0.14 M NaCl,2% casein, 0.5% BSA, 0.05% Tween 20, 0.1% NaN₃ [pH 7.3]) containing 20μg protein/ml of the E. coli extract and 100 μl of the urine sample wereadded. The mixture was stirred for 10 seconds and allowed to stand at37° C. for 1 hour. The wells were washed with 6 portions of PBST (0.05%Tween 20 and 0.05% NaN₃ in PBS) and 100 μl of a 11,000-fold dilution ofan enzyme (HRP)-labeled anti-human IgG antibody (peroxidase-conjugatedAffini Pure Goat Anti-human IgG (Fc), Jackson Immuno Research) in asecond buffer (50 mM Tris-HCl buffer, 0.14 M NaCl, 0.5% BSA, 5% goatserum, 0.05% Tween 20, 0.1% XL-II [pH 7.3]) was added. The plate wasallowed to stand at 37° C. for 1 hour and, then, washed with 6 portionsof PBST.

[0209] Then, 100 μl of a color developer solution (50 mMcitrate-Na₂HPO₄, 50% TMB solution, 0.0075% H₂O₂) was added and reactedat room temperature for 20 minutes, at the end of which time 100 μl of areaction stopper (50% TMB stop solution, 50% 1N—H₂SO₄) was added and theabsorbance was measured.

[0210] (5) Results

[0211] (i) In 5 cases of H. pylori-positive cases and the same number ofH. pylori negative cases as diagnosed by the ¹³C-UBT test [J.Gastroenterol., 33, pp.6-13, (1998)] which is regarded as the mostaccurate of all the diagnostic methods currently available for H. pyloriinfection, urine was sampled and the anti-H. pylori antibody in theurine was assayed by the procedure described under (4).

[0212] As a control experiment, the same procedure was applied to thesame urine samples except that the addition of the E. coli extract wasomitted and, based on the results, the effect of addition of an E. coliextract in accordance with the invention was evaluated.

[0213] The data are presented in FIG. 4.

[0214] In FIG. 4, the ordinate represents absorbance (O.D. 450˜650 nm)and the abscissa represents the addition (+) or omission (−) of the E.coli extract. Furthermore, the open circles represent the results onurine samples from H. pylori-positive patients by the ¹³C-UBT test andthe closed circles represent the results on urine samples from H.pylori-negative patients by the ¹³C-UBT test.

[0215] It will be apparent from FIG. 4 that by carrying out assays inthe presence of an E. coli extract in accordance with this invention, H.pylori-positive and −negative cases could be clearly discriminated inagreement with the results of the ¹³C-UBT test, endorsing the highaccuracy of the method of the invention.

[0216] (ii) Using urine samples from 99 healthy volunteers having nohistory of an eradication treatment against H. pylori and 20 patientswith stomach disease (7 cases of gastric ulcer and 13 cases ofgastritis), the urinary anti-H. pylori antibody was assayed in thepresence of the E. coli extract in accordance with the proceduredescribed under (4). The results are presented in FIG. 5. In FIG. 5, theordinate represents absorbance (O.D. 450 nm) and the abscissa representsthe groups according to the ¹³C-UBt test (positive and negative cases).The results indicated that, in the assay system containing an E. coliextract, all urine samples from negative patients gave definitelynegative results without a false positive result.

[0217] (iii) The amounts of anti-H. pylori antibody in sera from thesame subjects as enrolled in the experiment (ii) were determined withcommercial ELISA kits and the results were compared with the results ofassays in urine by the method of the invention in the same subjects inregard of sensitivity, specificity and accuracy. The serum and urinesamples from each subject were simultaneously collected and prepared forassays.

[0218] The commercial ELISA kits were as follows: HM-CAP™ kit, EntericProducts (HM-CAP); Helico G™ kit, Shield Diagnostic (Helico G); andHEL-p Test™ kit, Amrad Biotech (HEL-p). Each asay was performed inconformity with the protocol attached to each kit. The resutls are shownin Table 1. TABLE 1 Method of assay Sample Senmsitivity SpecificityAccuracy HM-CAP Serum 80% (56/70)  96% (47/49) 87% (103/119) Helico GSerum 99% (69/70)  88% (43/49) 94% (112/119) HEL-p Serum 97% (68/70) 90% (44/49) 94% (112/119) Method of Urine 99% (69/70) 100% (49/49) 99%(118/119) invention

[0219] Referring to Table 1, “Sensitivity” means the positive rategenerated with each kit in H. pylori-positive subjects(infection-positive according to ¹³C-UBT test: n=70), “Specificity”means the negative rate generated with each kit in H. Tylori-negativesubjects (infection−negative according to ¹³C-UBT test: n=49), and“Accuracy” means the percentage of accurate results with each kit in thetotal population (70+49=119 patients)

[0220] It is clear that despite the use of urine samples containing onlytrace amounts of the antibody, the determination of anti-H. pyloriantibody by the method of the invention gave higher detectionsensitivity and specificity as well as significantly higher accuracy ascompared with the conventional blood antibody assay kits.

[0221] (6) Assay of Anti H. pylori Antibody in Urine

[0222] Using a first buffer containing E. coli LPS (Difco) (LPSconcentration: 5 μg/well) in lieu of the E. coli extract, the procedureof (4) was otherwise repeated to determine the anti-H. pylori antibodyin urine samples and the results were evaluated in the same manner asunder (5) (i) . The results are presented in FIG. 6. It is clear fromthe diagram that similar results can be obtained by using E. coli LPS inlieu of said E. coli extract.

EXAMPLE 2 Assay of Anti-hepatitis B Virus (HBc) Antibody

[0223] (1) Assay of Anti-hepatitis B Virus (HBc) Antibody

[0224] An antigen plate was prepared using HBc antigen (ChemiconInternational) in accordance with the procedure of Example 1 (2) and theassay of anti-hepatitis B (core) (HBc) antibody in urine samples wascarried out in accordance with Example 1 (4).

[0225] Thus, 25 μl of first buffer containing the E. coli extract in avarying concentration and 100 μl of sample urine were added to each wellof the HBc antigen plate and after 10 seconds' stirring, the plate wasallowed to sit at 37° C. for 1 hour. After the plate was washed with 6portions of PBST, 100 μl of a 11,000-fold dilution of enzyme(HRP)-labeled anti -human IgG antibody in second buffer was added andthe plate was allowed to sit at 37° C. for 1 hour and then washed (6times with PBST).

[0226] Then, 100 μl of a color developer solution was added and thereaction was carried out at room temperature for 20 minutes, after which100 μl of a reaction stop solution was added and the absorbance wasmeasured.

[0227] (2) Results

[0228] In 5 positive and 5 negative blood anti-HBc antibody cases asclassified by assays using a commercial anti-HBc antibody assay kit(Dinabott), the anti-HBc antibody in urine was determined by theprocedure described under (1).

[0229] The concentrations of the E. coli extract in reaction mixtureswere set at 0, 0.78, 1.56, 3.13, 6.25, and 12.5 μg/ml and the effect ofaddition of the extract was evaluated. The results are presented in FIG.7.

[0230] In FIG. 7, the ordinate represents absorbance (O.D. 450˜650 nm)and the abscissa represents the level of addition of the E. coliextract. Further, the closed circles represent data on the urinaryantibody in patients with positive blood anti-HBc antibody and the opencircles represent data on the urinary antibody in patients with negativeblood anti-HBc antibody. It is apparent from the diagram that even whenurine samples are used, the difference in the detection level ofanti-HBc antibody between the group of patients with positive bloodanti-HBc antibody and the group of those with negative blood anti-HBcantibody becomes more prominent in relation to the level of addition ofthe E. coli extract.

EXAMPLE 3

[0231] Using urine samples which gave false positive tests in thedetermination of anti-HIV antibody in urine by a known assay method[Calypte™ HIV-1 Urine EIA: Arch. Pathol. Lab. Med., 119, 139-141 (1995);Clinical Infectious Diseases, 19, 1100-1104 (1994)], an exploratoryexperiment was carried out to identify the component supposedlyresponsible for a nonspecific reaction in the same assay system.

[0232] (1) Each of the above urine samples was adjusted to pH 7.4 with 1M phosphate buffer (pH 7.7) and filtered through 5.0, 0.8 and 0.2 μm-cutfilters. A 20 ml portion of the filtrate was concentrated byultrafiltration (a 10 kDa-cut membrane) to 2 ml. The concentrated urinewas subjected to gel permeation chromatography (Sephacryl S-300,Pharmacia) and each fraction was tested for its reactivity to HIVantigen.

[0233] The reactivity to HIV antigen was confirmed by causing eachfraction to react with an HIV antigen-immobilized plate prepared byimmobilizing HIV antigen (gp160) and detecting the conjugate(nonspecific binding component) with ALP-labeled goat anti-human(IgG+IgM) antibody, ALP-labeled goat anti-human IgG (Fc-specific)antibody or HRP-labeled goat anti-human IgG (Fab-specific) antibody (allavailable from Jackson ImmunoResearch Labs). The results arepresented-in FIG. 8.

[0234] In FIG. 8, the ordinate represents absorbance (O.D.) and theabscissa represents gel permeation chromatographic fractions (fractionNos.). The solid line represents the absorbance of the protein at 280 nmand the closed circle-line represents the result of detection with saidanti-human (IgG+IgM) antibody, the open triangle-line represents theresult of detection with said anti-human IgG (Fc-specific) antibody, andthe closed triangle-line represents the result of detection with saidanti-human IgG (Fab-specific) antibody.

[0235] It is apparent from FIG. 8 that in the detection with anti-humanIgG (Fab-specific) antibody, the mode of reaction (reactivity with thenonspecific binding component) is the same as in the detection withanti-human (IgG+IgM) antibody, while no reactivity is found withanti-human IgG (Fc-specific) antibody. This finding suggested that thenonspecific binding component is a fragment or denaturation product ofhuman IgG which retains the reactivity with anti-human IgG(Fab-specific) antibody without Fc region.

[0236] Therefore, it was clear that when an anti-human IgG (Fc-specific)antibody not reactive to such nonspecific binding component is used asthe assay reagent, the nonspecific reaction in the antibody detectionsystem and, hence, the incidence of a false positive test due to suchnonspecific reaction can be inhibited, with the result that a highlyspecific and very accurate antibody assay method can be provided.

EXAMPLE 4 Assay of Anti-HIV Antibody in Urine

[0237] To each well of an HIV antigen plate (Calypte™ HIV-I Urine EIA,Calypte Biomedical Corp.), 25 μl of first buffer (the sample buffer ofCalypte™ HIV-I Urine EIA) and 200 ρl of sample urine were added, andafter 10 seconds' stirring, the plate was allowed to sit at 37° C. for 1hour. After this plate was washed 6 times (wash buffer: D-PBS, 0.05%Tween 20), 100μ 1 of a 11,000-fold dilution of HRP-labeled goatanti-human IgG (Fc-specific) antibody (Peroxidase-conjugated Affini PureGoat anti-Human IgG, Fc Fragment Specific, Jackson ImmunoResearch Labs.)in second buffer (50 MM Tris-HCl buffer, 0.14M NaCl, 0.5% BSA, 5% GoatSerum, 0.05% Tween20, 0.1% XL-II (pH 7.3)) was added and the plate wasallowed to sit at 37° C. for 1 hour and washed (6 times) in the samemanner as above.

[0238] Then, 100 μl of a color developer (50% TMB solution, 50 mMCitrate-Na₂HPO₄, 0.0075% H₂O₂) was added and reacted at room temperaturefor 10 minutes, at the end of which time 100μ1 of a reaction stopsolution (50% TMB stopper, 50% 1N—H₂SO₄) was added and the absorbance(O.D. 450 nm) was measured.

[0239] As a control experiment, the sample was assayed with the knownassay method [Calypte™ HIV-1 Urine EIA: Arch. Pathol. Lab. Med., 119,139-141 (1995); Clinical Infectious Diseases, 19, 1100-1104 (1994)](control method) using ALP-labeled goat anti-human immunoglobulinantibody-as a second antibody. Furthermore, the negative control andpositive control of the above assay kit were measured by the above assaymethod of the invention. Since the absorbance values thus found werecomparable to those found with the above kit, the cut-off point for themethod of the invention was set at the value found by adding 0.180 tothe mean absorbance of the above negative control in accordance with thecut-off value calculation method of the same kit.

[0240] The results of assays in 100 samples (urine) from subjects withpositive serum anti-HIV antibody (2 cases) and subjects with negativeserum anti-HIV antibody (98 cases) are presented in Table 2. TABLE 2Method of invention Positive Negative Total Control Positive  4* 26 30method Negative 0 70 70 Total 4 96 100

[0241] It can be seen from Table 2 that although both the sensitivity ofthe method of the invention and that of the control method were 100%(2/2), the specificity was 71.4% (70/98) for the control method vs. 98%(96/98) for the method of the invention.

[0242] The above results indicate that as compared with the controlmethod, the antibody assay method of the invention is remarkably low inthe incidence of a false positive test and very high in specificity.

EXAMPLE 5 Assay of Anti-H. pylori Antibody in Urine

[0243] (1) Preparation of an H. pylori Antigen Plate

[0244] To a 100 mg/ml suspension of Helicobacter pylori (a clinicalisolate) in cold Dulbecco-PBS as prepared in the routine manner [J.Clin. Microbiol., 29: 2587-2589 (1991)], an equal volume of cold 0.2%Triton-X solution was added under constant stirring with a stirrer andthe mixture was further stirred for 5 minutes and centrifuged (3,000rpm, 20 min.). The supernatant was transferred to a new tube for use asthe extract (1˜1.5 mg/ml as protein).

[0245] This extract was diluted with D-PBS (2.5 μg/ml) and the dilutionwas distributed into a 96-well plate, 100 μl perwell, and incubated at25° C. overnight. After each well was washed, 300 μl of ablockingsolution (D-PBS, 0.5% casein, 5% sorbitol, 0.05% NaN₃ (pH 7.4)) wasadded, followed by incubation at 25° C. overnight. The blocking solutionwas then discarded and the plate was dried at 25° C. overnight, sealedtogether with a desiccant in an aluminum bag and stored at 40° C. untilused.

[0246] (2) Assays

[0247] 5 Using the immobilized antigen (plate) prepared as above,anti-H. pylori antibody in urine samples was assayed as in Example 4.

[0248] Thus, 25 μl of first buffer (200 mM Tris-HCl buffer, 0.14 M NaCl,2% casein, 0.5% BSA, 0.05% Tween 20, 0.1% NaN₃, 20 μg/ml E. coli extract(pH 7.3)) and 100 μl of sample urine was added to each well and after 10seconds' stirring, the plate was allowed to sit at 37° C. for 1 hour andthen washed 6 times.

[0249] Just as in Example 4, 100 μl of said dilution of HRP-labeled goatanti-human IgG (Fc-specific) antibody in second buffer was added and theplate was allowed to sit at 37° C. for 1 hour to detect the antibody.

[0250] The results obtained are presented in FIG. 9 and Table 3. TABLE 3Cut-off Sensitivity Specificity Accu- Sample Point ¹³C − UBT (+) ¹³C −UBT (−) racy Method Urine M + 3SD 56/56 (100%) 42/44 (95%) 98% of(0.104) inven- Urine M + 5SD 56/56 (100%) 43/44 (98%) 99% tion (0.147)Control Serum M + 3SD 53/56 (95%) 43/44 (98%) 96% method (1.27) SerumM + 5SD 47/56 (84%) 43/44 (98%) 90% (1.82)

[0251] In FIG. 9, the ordinate represents absorbance (O.D. 450˜650 nm)and the abscissa represents the positive H. pylori infection group (+;n=56) and negative H. pylori infection group (−; n=44) according to the¹³C-UBT test [J. Gastroenterol., 33: pp.6-13 (1998)].

[0252] In Table 3, “Sensitivity” denotes the percentage of casesdetected as positive among the positive cases according to ¹³C-UBT test;“Specificity” denotes the percentage of cases detected as negative amongthe negative cases according to ¹³C.-UBT test; and “Accuracy” denotesthe percentage of cases detected as positive and negative, respectively,among the positive and negative cases according to ¹³C-UBT test, therespective figures corresponding to the cut-off point of mean M+3SD orM+5SD. As a control experiment, the same urine samples were assayed witha serum anti-H. pylori antibody assay kit [HM-CAP; EPI/Kyowa Medics(K.K.)] and the results are also tabulated (Control method).

[0253] The above results indicate that the method of the invention issuperior to the control method in sensitivity and accuracy inparticular.

EXAMPLE 6 Assay of Anti-rubella Virus Antibody in Urine

[0254] (1) Preparation of a Rubella Antigen Plate

[0255] Using a commercial rubella antigen [available from BIO-DESIGN] ina concentration of 1 μg/ml and a blocking agent composed of D-PBS, 1%BSA, 5% sorbitol and 0.05% NaN₃ (pH 7.4) , the procedure of Example 5(1) was otherwise repeated to provide a rubella antigen plate.

[0256] (2) Assays

[0257] Using the above antigen plate, the rubella antibody in urinesamples was assayed in the same manner as in Example 5

[0258] (2). The Results are Presented in FIG. 10 and Table 4. TABLE 4Control method (serum ELISA) Positive Negative Total Method of inventionPositive 76 0 76 (urine ELISA) Negative 0 23 23 Total 76 23 99

[0259] In FIG. 10, the ordinate represents absorbance (O.D. 450˜650 nm)and the abscissa represents the serum anti-rubella antibody-positivegroup (n=76) and −negative group (n=23) according to the results ofdetermination with a commercial kit (Rubella IgG (II)-EIA, SEIKEN;available from Denka Seiken). The data given in Table 4 indicates acomplete agreement between the result obtained in urine by the method ofthe invention and the result obtained in sera by the control method.

[0260] According to the above data, the degree of agreement between themethod of the invention and the control method is as high as 100%(99/99), thus indicating that the invention enables detection of theantibody with high sensitivity and high specificity even in urine whichis safe and convenient and is, therefore, of great use in the laboratoryexamination.

EXAMPLE 7 Construction of an Antibody Assay Device

[0261] (1) Preparation of an H. pylori Antigen

[0262] An H. pylori antigen solution was prepared by the same procedureas in Example 1 (1) and stored at −80° C.

[0263] (2) Preparation of a Labeled Anti-human IgG Antibody-containingDry Glass Fiber

[0264] To a glass fiber sheet (5.0×260 mm×0.8 mm thick; Whatman) wasadded 1 ml of a 40 nm (dia.) colloidal gold-labeled anti-human IgG(Fc-specific) antibody solution and the sheet was dried overnight. Thissheet was stored together with a desiccant at room temperature untilused.

[0265] (3) Preparation of a Membrane

[0266] The H. pylori antigen solution (3 mg/ml) prepared under (1) aboveand an anti-human IgG antibody solution (0.3 mg/ml) were respectivelyapplied onto a nitrocellulose membrane (26.5 mm×260 mm×0.1 mm thick;Advance MicroDevice) by spraying (1.5 μl/cm) in lines at a predeterminedspacing as illustrated in FIG. 11 and dried at 37° C. for 120 minutes.After drying, the membrane was dipped and washed in a skimmilk-containing Borax Buffer (pH 8.2) for 30 minutes. The washedmembrane was dried at 37° C. for 1 hour and stored in the presence of adesiccant at room temperature.

[0267] (4) Assembly (a Solid-phase Support)

[0268] As illustrated in FIG. 3 (A), the above membrane (3), anabsorbent filter paper pad (4) (22×260 mm×1.5 mm thick; Whatman), saidlabeled antibody-containing glass fiber sheet (2) and a sample pad (1)(15×260 mm×1.0 mm thick; filter paper, Whatman) were glued together withan adhesive and cut to 5 mm in width.

[0269] The solid-phase support thus prepared was set in position on abottom section (8) of a plastic housing and a top section (7) providedwith a sample inlet port (9) and a detection window (10) in series wasplaced over the solid-phase support and set securely on the bottomsection (8).

EXAMPLE 8 Assay of Anti-H. pylori Antibody in Urine

[0270] Using the device constructed in Example 7, the assay of anti-H.pylori antibody was carried out in 3 kinds of urine samples, namelysamples of urine from subjects with H. pylori infection, samples ofurine from subjects without H. pylori infection, and extremely leansamples of urine from subjects with H. pylori infection.

[0271] First, 500 μl of sample urine was added to 500 μl of a samplediluent [200 mM Tris-HCl buffer, 0.14MNaCl, 2% casein, 0.5% BSA, 0.05%Tween20, 0.1% NaN₃ (pH7.3), E.coli LPS (Difco) 50 μl/ml], followed bymixing. Six drops (about 150 μl) of the resulting dilution was drippedfrom the sample inlet port (9) of the device constructed in Example 7for adsorption on the support which was then allowed to sit for 20minutes. As a result, when a qualified urine sample was used, a pink˜redcolor band appeared in the control region of the detection window (10).On the other hand, when an extremely lean unqualified urine sample wasused, neither the test region nor the control region of the detectionwindow (10) showed a color development, indicating that the sample wasnot evaluable. When the sample was a qualified urine sample from asubject without H. pylori infection, a pink˜red color band appeared onlyin the control region of the detection window (10) showing a negative(true negative) test for H. pylori infection, while in the presence ofH. pylori infection, a pink˜red color band appeared in both the testregion and control region of the detection window (10), showing apositive test for H. pylori.

EXAMPLE 9 Assay of Anti-H. pylori Antibody in Urine

[0272] (1) Preparation of an E. coli Component

[0273]Escherichia coli (pvc18/JM109; Takara Shuzo) was cultured inampicillin-containing liquid LB medium (Luria-Bertani medium; NihonSeiyaku) at 37° C. for 18 hours and the grown cells were harvested bycentrifugation and washed with 2 portions of PBS. Then, cold PBS wasadded at a final cell concentration of 100 mg/ml and the cells weredisrupted and extracted using a sonicator (10 seconds×3 times). Theresultant supernatant was used as E. coli extract protein. (2) Using theE. coli extract protein prepared under (1) above in lieu of the E. coliLPS added to the sample diluent in Example 8, the procedure of Example 8was otherwise repeated to determine anti-H. pylori antibodies in urinesamples. As a result, the same results as described in Example 8 wereobtained.

EXAMPLE 10

[0274] Using the whole blood, plasma and urine from 21 H. pyloriinfection-positive subjects and the same number of H. pyloriinfection−negative subjects (a total of 42 cases) according tothe¹³C-UBT test [J. Gastroenterol., 33:6-13 (1998)], anti-H. pyloriantibodies in samples were assayed by the procedure described in Example8.

[0275] As a control experiment, samples from the same subjects wererespectively assayed using commercial H. pylori antibody assay kitsdirected to whole blood or plasma and the effectiveness of the device ofthe invention was evaluated from the data. The results are presented inFIG. 12.

[0276] In FIG. 12, control kits A through H were as follows.

[0277] A: Helitest (manufactured by Cortecs Diagnostics)

[0278] B: H. pylori-Check-1

[0279] (Manufactured by Bio-Medical Products)

[0280] C: First Check H. pylori

[0281] (Manufactured by Worldwide Medical Corp)

[0282] D: Biocard Helicobacter pylori IgG

[0283] (Manufactured by Anti Biotech Oy)

[0284] E: Insta Test H. pylori

[0285] (Manufactured by Cortez Diagnostics Inc.)

[0286] F: One Step H. pylori Test

[0287] (Manufactured by Teco Diagnostics)

[0288] G: H. pylori SPOT

[0289] (Manufactured by International Immuno-Diagnostics)

[0290] H: Quick Stripe H. pylori

[0291] (Manufactured by Diatech Diagnostics Inc.)

[0292] In FIG. 12, “Specificity” denotes the percentage of negativetests (negative rate) as found by assaying ¹³C-UBT test-negative sampleswith the corresponding kit, and “Sensitivity” denotes the percentage ofpositive tests (positive rate) as found by assaying ¹³C-UBT-positivesamples with the corresponding kit.

[0293] It is apparent from the data in FIG. 12 that the antibody assaydevice and solid-phase assay method of the-present invention provideexcellent assay systems with high detection specificity and accuracyeven when applied to urine samples, not to speak of blood (whole blood,plasma) samples.

[0294] It is also clear from the above results that even when the sampleis a urine sample which is safe and convenient, the present inventionenables high-sensitivity, high-specificity detection of antibodies, thusbeing of great use in the laboratory examination.

EXAMPLE 11 Effect of an E. coli Component on the Assay of Antibodies inUrine

[0295] (1) The effect of an E. coli component on the assay of antibodiesin urine was evaluated using the assay device constructed in Example 8.Thus, using the urine from H. pylori infection-positive and −negativesubjects selected by the ¹³C-UBT test, anti-H. pylori antibodies inurine samples were assayed in a system using a sample diluent notcontaining E. coli LPS (Diluent 1) and systems using the same diluentsupplemented with E. coli LPS at the various concentrations shown inTable 5. The line color development in the test region and controlregion was evaluated from the line intensity measured with adensitometer (manufactured by ATTO). The results are shown in Table 5.TABLE 5 Level of addition of E. coli LPS (μg/ml) Diluent 1 100 33.3 11.13.7 1.2 Positive Control site 50 51 43 36 41 45 urine Test site 68 66 6261 61 67 Negative Control site 26 26 22 18 23 23 urine Test site 12 0 00 13 12

[0296] It was found that when E. coli LPS was added to the sample at11.1 μg/ml and higher levels, the nonspecific reaction observed withDiluent 1 disappeared so that a false positive test (detection error)could be precluded.

Industrial Applicability

[0297] The present invention provides an antibody assay technology bywhich target antibodies specific to sources of infection can be detectedwith high sensitivity and high specificity even when urine samples whichare comparatively lean in the antibodies are used as test samples.According to the antibody assay method of the invention, the “falsepositive” reactions due to contaminants in samples can be significantlyinhibited so that highly accurate and dependable assay results can beobtained. Moreover, the present invention provides improvements inimmunocapillary or inmmunochromatographic assays, whereby the existenceof target antibodies and their amounts in samples can be detectedaccurately with a clear distinction between “false negative” and “truenegative”.

1. A method for assaying an antibody comprising detecting a targetantibody in a sample by utilizing an antigen-antibody reaction,characterized in that said antigen-antibody reaction is carried outbetween said antibody and an assay antigen in the presence of an E. colicomponent.
 2. The antibody assay method according to claim 1 whereinsaid E. coli component is at least one member selected from the groupconsisting of the soluble fraction and lipopolysaccharide fraction of E.coli.
 3. The antibody assay method according to claim 1 wherein said E.coli component is used in a proportion of about 0.1˜100 μg per μg of theassay antigen.
 4. A method for assaying an antibody comprising detectinga target antibody in a sample by the sandwich technique, characterizedin that a substance having a specific affinity for the Fc region of thetarget antibody IgG is used as an antibody assay reagent.
 5. Theantibody assay method according to claim 4 wherein the antibody assayreagent is an Fc-specific anti-IgG antibody.
 6. The antibody assaymethod according to claim 4 which comprises an antigen-antibody reactionstep in which the target antibody in the sample is coupled to animmobilized form of an antigen to said antibody as immobilized on asupport and a reaction step in which the target antibody captured bysaid immobilized antigen is reacted with an antibody assay reagenthaving a specific affinity for the Fc region of the antibody IgG.
 7. Theantibody assay method according to claim 6 wherein the antigen-antibodyreaction is carried out in the presence of an E. coli component.
 8. Theantibody assay method according to claim 1 or 4 wherein the sample is aurine sample.
 9. The antibody assay method according to claim 1 or 4wherein the target antibody is the antibody against a source ofinfection.
 10. The antibody assay method according to claim 1 or 4wherein the source of infection is a member selected from the groupconsisting of human immunodeficiency virus, hepatitis viruses, rubellavirus, influenza virus, measles virus, herpes virus, cytomegalovirus,Clamydia spp., gonococci, Helicobacter pylori and Toxoplasma gondii. 11.The antibody assay method according to claim 1 or 4 wherein the antigenis a member selected from the group consisting of bacteria, viruses,protozoae and components of bacteria, viruses or protozoae which containat least the antigenic determinant groups of said bacteria, viruses orprotozoae.
 12. The antibody assay method according to claim 1 or 4wherein the antigen is the microorganism Helicobacter pylori or acomponent thereof which contains at least the antigenic determinantgroup of said microorganism.
 13. An antibody assay device comprising asolid phase support having at least (a) a first region to which a sampleis applied and (b) a second region in which the antibody in the testsample is reacted as arranged in such a sequence that the sample istransported from the first region to the second region by capillaryaction, and a labeling means for detecting the result of reaction in thesecond region, said (b) second region having (i) a test site where aligand for capturing the target antibody to be detected has beenimmobilized and (ii) a control site where a ligand for capturing anarbitrary antibody in the sample has been immobilized.
 14. The antibodyassay device according to claim 13 wherein the ligand immobilized in thetest site is an antigen to the target antibody occurring in the sample.15. The antibody assay device according to claim 13 wherein the ligandimmobilized in the control site is an anti-human immunoglobulin antibodycapable of capturing an arbitrary antibody in the sample.
 16. Theantibody assay device according to claim 13 comprising a labeled ligandto be bound by both the target antibody and arbitrary antibody as saidlabeling means.
 17. The antibody assay device according to claim 13wherein the labeling means is a labeled ligand to be bound by both thetarget antibody and arbitrary antibody as removably supported upstreamsof the second region of the solid phase support in such a manner that,upon contact with a sample, it reacts with the target antibody andarbitrary antibody to form a target antibody/labeled ligand complex andan arbitrary antibody/labeled ligand complex, respectively, which arethen transported by capillary action to the second region where they arefixed in the test site and control site, respectively.
 18. The antibodyassay device according to claim 17 wherein the labeled ligand issupported in a region (tracer region) intermediate between the firstregion and second region of the solid phase support.
 19. The antibodyassay device according to claim 17 wherein the labeled ligand to bebound by both the target antibody and arbitrary antibody is a labeledanti-human immonoglobulin antibody.
 20. The antibody assay deviceaccording to claim 19 wherein the anti-human immunoglobulin antibody isan anti-IgG antibody having a specific affinity for the Fc region ofimmunoglobulin G.
 21. The antibody assay device according to claim 13wherein the solid phase support is further provided with an absorptionregion downstreams of the first and second regions so that the sampletransported from the first region to the second region is furthertransported by capillary action to the absorption region.
 22. Theantibody assay device according to claim 13 wherein the couplingreaction of the target antibody at the test site in the second regiontakes place in the presence of an E. coli component.
 23. The antibodyassay device according to claim 13 wherein the sample is a urine sample.24. Use of the antibody assay device claimed in any of claims 13 through22 for assay of a target antibody directed to a source of infection asoccurring in a sample.
 25. The use according to claim 24 wherein thesample is a urine sample.
 26. A method for solid phase assay of a targetantibody in a sample which comprises applying the sample to the firstregion of the antibody assay device claimed in claim 13 and detectingthe development of a color at the test site in the second region underthe condition of the control site in the second region developing acolor.
 27. A method for solid phase assay of a target antibody in asample which comprises applying the sample to the first region of theantibody assay device claimed in claim 20 and detecting the developmentof a color at the test site in the second region under the condition ofthe control site in the second region developing a color.
 28. The methodfor solid phase assay of a target antibody according to claim 26 or 27wherein the coupling reaction of the target antibody at the test site inthe second region of the antibody assay device takes place in thepresence of an E. coli component.
 29. The method for solid phase assayof a target antibody according to claim 26 wherein the sample is a urinesample.
 30. An antibody assay reagent kit for use in assaying anantibody in a sample by utilizing an antigen-antibody reaction,characterized in that said antibody assay reagent kit contains an E.coli component.
 31. An antibody assay reagent kit characterized by itscontaining a substance having a specific affinity for the Fc region ofthe target antibody IgG as an antibody assay reagent.
 32. An antibodyassay reagent kit containing the antibody assay device claimed in claim13.